Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics

Abstract

We have used the site-directed spectroscopies of time-resolved fluorescence resonance energy transfer (TR-FRET) and double electronelectron resonance (DEER), combined with complementary molecular dynamics (MD) simulations, to resolve the structure and dynamics of cardiac myosin-binding protein C (cMyBP-C), focusing on the N-terminal region. The results have implications for the role of this protein in myocardial contraction, with particular relevance to β-adrenergic signaling, heart failure, and hypertrophic cardiomyopathy. N-terminal cMyBP-C domains C0-C2 (C0C2) contain binding regions for potential interactions with both thick and thin filaments. Phosphorylation by PKA in the MyBP-C motif regulates these binding interactions. Our spectroscopic assays detect distances between pairs of site-directed probes on cMyBP-C. We engineered intramolecular pairs of labeling sites within cMyBP-C to measure, with high resolution, the distance and disorder in the protein's flexible regions using TR-FRET and DEER. Phosphorylation reduced the level of molecular disorder and the distribution of C0C2 intramolecular distances became more compact, with probes flanking either the motif between C1 and C2 or the Pro/Ala-rich linker (PAL) between C0 and C1. Further insight was obtained from microsecond MD simulations, which revealed a large structural change in the disordered motif region in which phosphorylation unmasks the surface of a series of residues on a stable α-helix within the motif with high potential as a protein-protein interaction site. These experimental and computational findings elucidate structural transitions in the flexible and dynamic portions of cMyBP-C, providing previously unidentified molecular insight into the modulatory role of this protein in cardiac muscle contractility. muscle | protein kinase A | fluorescence resonance energy transfer | DEER | molecular dynamics simulation

Cardiac myosin-binding protein C: A protein once at loose ends finds its regulatory groove

“…The present studies by Previs et al (11) and Colson et al (12) establish a compelling yet challenging framework for understanding the molecular mechanisms of regulation via phosphorylation of cMyBP-C. Using distinct experimental approaches, the studies have impressive similarities in demonstrating structural changes, i.e., reductions in both length and disorder, in the regulatory M domain near the N terminus of cMyBP-C due to phosphorylation of the three (or four) serines within the domain.…”

“…Using complementary cellfree approaches, two reports (11,12) in PNAS present exquisite insights into the structural effects of phosphorylation on N-terminal regions of cMyBP-C. These results not only suggest specific mechanisms of the protein's function in vivo but also give rise to new questions that promise to drive research for years to come.…”

High-Throughput Spectral and Lifetime-Based FRET Screening in Living Cells to Identify Small-Molecule Effectors of SERCA

“…The high-resolution FRET approach, coupled to functional assays, is applicable to a wide range of protein targets, including the ryanodine receptor 29 , myosin 18,30 , phospholamban 10 , multiple-drug resistance receptor 31 , and the tumor necrosis receptor 32 . The ability to quickly and reliably assess structural perturbations from biosensors in relation to physiologically-relevant functional changes holds high promise for the development of allosteric effectors and potentially valuable lead compounds.…”

The contribution of modern EPR to structural biology

“…Based on the Anfinsen dogma, it became customary to explain function in terms of a single conformation or of well-defined transitions between a few conformations defined at atomic resolution. While this is certainly a reasonable approximation in some cases [75][76][77], availability of distance distributions demonstrates that rather often conformation transitions are coupled to order-disorder transitions or are shifts in disorder equilibria [39,[78][79][80][81][82][83][84][85][86][87][88][89][90][91][92][93][94]. Among the systems addressed by PDS to date, the fraction where at least one state is genuinely disordered is surprisingly large.…”

“…Synaptotagmin 1 remains heterogeneous after binding to SNAREs [78]. In cardiac myosin-binding protein C, phosphorylation causes compaction and reduces disorder of the Pro/Ala-rich linker between two immunoglobin domains [88]. The human and teleost fish secretory components, which could be crystallized, exhibit well-defined conformations in the absence of their immunoglobulin ligands [95], whereas the unliganded avian secretory component is flexible, pointing to a distinct mechanism of ligand binding of secretory components among vertebrates [91].…”